Çukurova bölgesi pamuk alanlarında Aphis gossypii (Glover)’da aşırı düzeyde sitokrom P450 genleri ekspresyonu
RESULTS AND DISCUSSION
Enzyme assays and gene expression analyses were carried out on A. gossypii populations that were collected from different locations in the province of Adana. As a result of the analyses, higher levels of AChE, GSTs and cytochrome P450 activities were observed in all the collected populations in comparison not the reference population. The highest AChE activity was seen in the Çiftlikler population (0.82 U/
ml), while this was followed by the Hamitbey population (0.62 U/ml). The lowest activity was observed in the Akyuva population (0.34 U/ml). 5.9 times more AChE activity was observed in comparison to the reference population (Table
1). In parallel with these results, another study that was carried out in this region found 6.5 times more AChE activity in comparison to the reference population for A. gossypii populations (Ulusoy et al. 2018, Velioglu et al. 2008). As in the cases of AChE enzyme activity levels, the highest GST enzyme activities were found in the Çiftlikler population (2.68 U/ml) and Hamitbey population (2.33 U/ml). 4.3 times higher GST enzyme activity was observed in comparison to the reference population. The weakest GST activity was found in the Akyuva population (1.05 U/ml) (Table 1). Likewise, it was reported that, 3.3 times more enzyme activity was found in the neonicotinoid-resistant A. gossypii populations in the province of Adana in 2016 (Ulusoy et al.
2018). These results provide evidence that these populations have metabolic resistance still in this region. Previous studies reported that the general levels of esterase and GST activities may be higher in populations that are resistant against the insecticides in the groups of organophosphates, carbamates and pyrethroids (Devonshire and Moores 1982, Hemingway and Georghiou 1984, Rauch and Nauen 2003). Especially the Çukurova region is an agricultural production area where there is a widespread usage of insecticides from different groups in the agricultural fields where polyculture agriculture activities including vegetables, fruits and cotton are carried out. Although different agricultural products are grown, these agricultural fields are almost intertwined. Therefore, considering that A. gossypii is a polyphagous species and is exposed to substantial amounts of insecticides with different effect mechanisms such as organophosphates, carbamates, pyrethroids and neonicotinoids, it is inevitable to consider a metabolic resistance. The observation of cytochrome P450 enzyme activities revealed the highest enzyme activity in the Hamitbey population (0.74 U/ml) followed by the Solaklı population (0.64 U/ml), while the lowest activity was found in the Körkuyu population (0.29 U/ml). The Hamitbey population showed 4.3 times higher enzyme activity in comparison to the reference population (Table 1). A similar study that was carried out in this region also reported higher cytochrome P450 enzyme activity (Ulusoy et al. 2018).
In the study, RT-PCR analysis was carried out to observe A. gossypii’s cytochrome P450 CYP6CY13, CYP6CY22 and ß-actin genes in agarose-gel (Figure 1). When the gene expression of cytochrome P450 CYP6CY13 and CYP6CY22 were analyzed by RT-PCR, higher relative activities were recorded in comparison to the reference population (Figure 2). The relative activity levels of the CYP6CY22 gene were 81.9 higher in the Hamitbey population and 46.3 times higher in the Solaklı population. The highest relative activity level of the CYP6CY13 gene was found in the Gazipaşa population (6 times) (Figure 2). The relative activity levels of both genes
were found to be higher in comparison to the reference.
Hirata et al. (2017) reported 45 times higher relative activity in the CYP6CY13 and CYP6CY22 genes of thiamethoxam-resistant A. gossypii individuals in comparison to the reference individuals. The finding of a study that was carried out in 2016 in this region on the presence of thiamethoxam-resistant A. gossypii populations supports the finding of high relative activity levels (Ulusoy et al. 2018). Previous studies conducted in the Eastern Mediterranean Region reported resistance against neonicotinoids in A. gossypii in the cotton production fields especially in the year 2016 (Ulusoy et al.
2018). Previous studies stated that especially the activity of the P450 enzyme group was effective in the development of such resistance (Rauch and Nauen 2003, Scott 1999, Zhao et al. 2014). Additionally, studies have demonstrated that, as well as the presence of mutations in resistance against neonicotinoid insecticides, the metabolic activity and gene Figure 2. Cytocrome P450 CYP6CY13 and CYP6CY22 genes relative expression levels of Aphis gossypii populations (R/S: resistant population/susceptiple population)
Figure 1. Aphis gossypii CYP6CY13, CYP6CY22 and ß-actin genes RT PCR agarose gel electrophrosesis photograph.
M: 100 bp ladder, 1. Negative control, 2. ß-actin: 60 bp, 3.
CYP6CY13: 234 bp 4. CYP6CY22: 326 bp
expressions of the P450 group were also higher (Hirata et al.
2017, Nauen et al. 2008, Wang et al. 2009, Wu et al. 2018).
While the CYP4, -6, -9, and -12 gene family in insects plays a role in the biological detoxification process, it was reported that the CYP4 and CYP6 subfamilies were the most effective members in the xenobiotic mechanism and observation of resistance (Berenbaum 2002, Li et al. 2007). Consequently, this study showed that the samples that were collected from locations of the cotton production fields in the province of Adana in 2018 were highly exposed to insecticides, and there was an increased metabolic resistance. The high levels of the AChE, GST and cytochrome P450 enzyme activities that were observed in the collected A. gossypii populations supported the development of resistance. Additionally, as shown by the total RNA and RT-PCR analyses, in parallel with cytochrome P450 activity, the expression levels of the CYP6CY13 and CYP6CY22 genes were also higher. The presence of increased metabolic resistance was observed in an accelerating manner considering the results of previous studies on resistance against insecticides in the neonicotinoid group in the Çukurova region. It was also revealed that there might be a development of resistance caused by cytochrome P450.
These days, the discovery and development of new insecticides is very difficult and costly task. Therefore, strategies that slow down the rapid development of resistance to new and existing insecticides need to be implemented in all agricultural environments (Sparks and Nauen 2015). If insecticide resistance management (IRM) is not seriously taken into account in pest control, the development of resistance will be inevitable. The basis of an IRM strategy is
reduce selection pressure and avoiding selection of resistance mechanisms (Bielza 2008). The repeated use of the same insecticide compounds of the same modes of action over many generations is cause insecticide resistance development.
An Integrated Pest Management (IPM) strategies, cultural control (proper watering and fertilization, sanitation, weed removal, crop rotation), biological control (use of predators, parasitoids and pathogens), and genetic control (host plant resistance) should not be ignored (Biondi et al. 2018, Guedes and Picanço 2012). The use of one or more of these alternative strategies may reduce the need for insecticides, thus decreasing the selection pressure on pest population. In the light of these results, it was shown that the insecticide-based control against A. gossypii should be reevaluated in the cotton field in the Çukurova region. Consequently, in the management of A. gosyypii, it is necessary to take a more conscious and serious approach by considering the IRM and IPM strategies again.
ACKNOWLEDGEMENTS
Part of this work was published as oral presentation at the IV.
International Agriculture Congress (5-8 July 2018) Nevşehir, Turkey.
ÖZET
Polifag bir tür olan Aphis gossypii Glover, 1877 (Hemiptera:
Aphididae) tüm dünyada pamuk bitkisinin ana zararlılarındandır. Çukurova bölgesi polikültür tarımın yapıldığı bir bölgedir. Bu bölgede pamuk ekim alanlarında A. gossypii mücadelesinde neonikotinoid grubu insektisitler yoğun bir şekilde kullanılmakta olup, direnç problemi ile
Population AChE U/ml GSTs U/ml Cytochrome P450
U/ml
Gazipaşa 0.52 ± 0.04c 1.63±0.73bc 0.45±0.12bcd
Solaklı 0.57 ± 0.08cd 1.72±0.38bc 0.64±0.14de
Körkuyu 0.45± 0.12bc 1.40±0.7b 0.29±0.12ab
Akyuva 0.34±0.06b 1.05±0.06b 0.52±0.07cd
Çiftlikler 0.82±0.05e 2.68±0.28d 0.39±0.06bc
Hamitbey 0.67±0.02 d 2.33±0.33cd 0.74±0.14e
Susceptible 0.14±0.05 a 0.63±0.06a 0.15±0.12a
Table 1. Acetylcholin esterase (AChE), glutathione S-transferase (GTS), cytochrome P450 monooxygenase (P450) enzyme activities of resistant and susceptible A. gossypii populations collected from Cukurova region
a, b, c, d,e – Duncan’s multiple range test (p< 0.05)
karşılaşılmaktadır. Çalışmada 2018 yılı, Adana ili pamuk alanlarından toplanmış altı farklı A. gossypii popülasyonu sitokrom P450 (CYP)’ye ait CYP6CY22 ile CYP6CY13 genleri relatif ekspresyon düzeyleri, sitokrom P450, asetilkolin esteraz (AChE) ve glutathion S-transferaz (GST) enzim etkinlikleri belirlenmiştir. Hassasa oranla Hamitbey popülasyonunda CYP6CY22 geni 81.9 kat ve Gazipaşa popülasyonunda CYP6CY13 geni 6 kat daha fazla gen regülasyonu belirlenmiştir. Ayrıca Hamitbey popülasyonunda 4.3 (0.74 U/ml) kat daha fazla sitokrom P450 enzim aktivitesi saptanmıştır. Çiftlikler popülasyonunda 5.9 kat AChE (0.82 U/ml) ve 4.3 kat daha fazla GST (2.68 U/
ml) aktivitesi gözlenmiştir. Yapılan analizlerde A. gosyypii popülasyonlarının yoğun şekilde insektisitlere maruz kaldığı saptanmıştır. Sonuç olarak, aşırı gen ekspresyonu ve yüksek düzeydeki enzim etkinlikleri, sitokrom P450 aktivitesine dayanan metabolik direncin varlığını ortaya koymuştur.
Anahtar kelimeler: Aphis gossypii, pamuk, sitokrom P450, aşırı ekspresyon
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Cite this article: Ulusoy, S. (2020). Overexpression of cytochrome P450 genes in Aphis gossypii (Glover) in the cotton fields of the Çukurova region, Turkey. Plant Protection Bulletin, 60-1. DOI: 10.16955/bitkorb.540632
Atıf için: Ulusoy, S . (2020). Çukurova bölgesi pamuk alanlarında Aphis gossypii (Glover)’da aşırı düzeyde sitokrom P450 genleri ekspresyonu. Bitki Koruma Bülteni, 60-1. DOI:
10.16955/bitkorb.540632
Original article